Multiple coordinate controls contribute to a balanced expression of ribulose-1,5-bisphosphate carboxylase/oxygenase subunits in rye leaves.
In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose-1,5-bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment-specific inhibitors, or by the use of 70S-ribosome-deficient leaves. The latter were generated by growing the plants at a non-permissive elevated temperature of 32 degrees C. The rates of synthesis of the two ribulose-1,5-bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5-2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S-ribosome-deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S-ribosome-deficient leaves after application of an inhibitor of cytoplasmic translation. MDMP [2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S-ribosome-deficient leaves were degraded with a half-time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The proteolytic removal of surplus free small subunits is regarded as a final post-translational fine-tuning step to establish a balanced subunit stoichiometry in leaves.
Winter
U
U
Botanisches Institut, J. W. Goethe-Universität, Frankfurt am Main, Federal Republic of Germany.
Feierabend
J
J
D000596:Amino Acids / Q000378:metabolism; D002474:Cell-Free System; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D003593:Cytoplasm / Q000201:enzymology; D002523:Edible Grain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000528:radiation effects; D015151:Immunoblotting; D008027:Light; D008715:Methionine / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D011422:Propionates / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000528:radiation effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism* / Q000502:physiology; D012335:RNA, Ribosomal / Q000502:physiology; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis* / Q000235:genetics; D012434:Secale / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
D000596:Amino Acids; D010455:Peptides; D011422:Propionates; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012335:RNA, Ribosomal; C002167:2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide; D008715:Methionine; D012273:Ribulose-Bisphosphate Carboxylase